Study on expression of transcription factors AP2-Domain, HD-ZIP, WRKY and MYB in oily sunflower (Helianthus annuus L.) under drought stress

Sunflower (Helianthuse annuus L.) is an annual plant from Composite with a chromosome number of 2n = 2x = 34 which is widely cultivated for supplying edible oil. Drought is one of the most important environmental stresses that limits the growth and distribution of plant more than other factors. This plant is classified as semi-tolerant to drought stress; however, its performance is negatively affected by drought. Transcription factors are molecules that play an important role in the understanding and transmission of stress messages as well as many physiological processes. One of the most effective ways to deal with stress is to produce resistant hybrids. Investigation and study of expression of genes post stress application and identification of genes involved in resistance and especially regulatory genes such as transcription factors is vital and necessary for molecular breeding programs.

In order to investigate the effect of drought stress on the expression of transcription factors: AP2-Domain, HD-ZIP, WRKY and MYB in oilseed sunflower, two lines with different susceptibility to drought stress were selected and cultivated in a completely randomized design with three replications in greenhouse. The seeds were planted in 3 cm depth of 30 × 25 cm pots containing farm soil and sand mixture in the ratio of 2:1. The plants were grown in controlled conditions at 25 ± 3 °C, 65% relative humidity and 12 h dark-light photoperiod and were irrigated regularly at 100% of field capacity up to 8-leaf stage. After this stage, a number of pots were kept at the same field capacity however, some other were exposed to 80, 60 and 40% of field capacity. Samplings were done in two times, one and three weeks after drought stress application. The study of the expression of genes was performed using real time PCR by SYBR Green method. RNA extraction kit RNX-plusTM (Sinoclon Co., Iran) and complementary DNA (cDNA) synthesis Kit (Fermentas LIFE SCIENCE # K1621) were used according to the manufacturer's protocols. Quantitative reverse transcription-PCR (qRT-PCR) was performed in triplet using 6.25 μl of Maxima SYBR Green/ Fluorescein qPCR Master Mix (2X) (Thermo Fisher Scientific, Germany), 5 pM of forward and reverse primers and 50 ng of cDNA for each reaction in a final volume of 12.5μl. Relative gene expression was analyzed by comparative Ct method, 2−ΔΔC. Target gene was normalized by the reference gene, ACTIN and calibrated for each sample against the control.

The results of statistical analyzes showed that the expression of the genes in the susceptible and resistant lines of sunflower is different. Mean comparisons of expression of AP2-Domain, WRKY and MYB transcription factors in the two genotypes ENSAT254 (tolerant) and LC1064C (susceptible) showed that the expression level was not tangible in the first week after drought stress application, but the expression of genes was increased in 40% of field capacity in the third week post drought stress application especially in ENSAT254 genotype. In relation to HD-ZIP transcription factor, the expression was much higher in ENSAT254 genotype than LC1064C genotype in the first week of sampling at 40% stress intensity. In the third week of sampling, the expression level of both genotypes increased in 40% of field capacity, although the expression was slightly higher in LC1064C genotype.

Early expression of HD-ZIP transcription factor appears to be involved in increasing genotype resistance to drought stress. The results of the present study can be useful in sunflower improvements programs for producing and developing drought tolerant cultivars.